The heart has a remarkable ability to change the extent to which myocytes are electrically coupled by remodeling gap junctions in response to changing physiologic and pathophysiologic conditions. Regulation of the formation and function of gap junction channels is, therefore, of critical importance in myocardial function during health and disease. To gain mechanistic insights into the regulation of intercellular coupling in the cardiac hypertrophic response, an in vitro model of pulsatile mechanical stretch of cultured myocytes was designed to simulate overload in the heart in vivo. Only one hour of stretch leads to a 2-fold increase in Cx43 expression and a 30% increase in impulse propagation velocity. This experimental system will be exploited to elucidate cellular mechanisms and signaling pathways regulating intercellular coupling during the hypertrophic response. We propose that stretch induces upregulation of Cx43 through multiple signaling pathways that have different time courses of action and work through different cellular mechanisms. The early response to stretch occurs within 15 minutes and induces upregulation of Cx43 expression by alterations in Cx43 trafficking and degradation mediated by vascular endothelial growth factor signaling. The later response to stretch (>1-2 hours) is mediated by angiotensin II signaling and contributes to the upregulation of Cx43 expression by increasing Cx43 synthesis. In Specific Aim 1, the time course of stretch-induced upregulation of Cx43 will be defined and the relative contributions of alterations in Cx43 trafficking, synthesis and degradation will be delineated. In Specific Aim 2, the temporal relationships of stretch-activated signaling pathways initiated by vascular endothelial growth factor and angiotensin II in regulating changes in Cx43 trafficking, synthesis and degradation will be determined. In Specific Aim 3, the role of alterations in Cx43 phosphorylation at specific tyrosine residues in stretch-induced upregulation of Cx43 expression will be defined. Elucidation of mechanisms enhancing intercellular coupling will provide the potential for novel therapeutic strategies for the treatment of heart failure and prevention of arrhythmias.